The present invention relates to a DNA analyzing method including DNA sequencing.
Conventional DNA sequencing of large segments of DNA (e.g. 10.sup.5 to 10.sup.6 (100 Kb to 1 Mb) base length) requires subcloning processes. During such subcloning processes, target DNA is digested with restriction enzymes to produce fragments of the target DNA. The fragments are cloned in a plasmid, which is used as a vector in the cloning process. E. Coli transformed with the plasmid is cultured in agar thereafter. As only transformed E. Coli can survive and form colonies in the agar, we can select E. Coli transformed by the plasmid by picking the colonies. Then the target DNA fragments subcloned in the plasmid are isolated from E. Coli and the isolated target DNA fragments are sequenced. The target DNA fragments are then separated by picking obtained colonies or clones and DNA sequencing carried out for each clone of the plasmid containing the isolated DNA fragment. Normally, 300 to 500 DNA bases can be determined by one sequencing operation, and sequencing of 1 mega (10.sup.6) base requires analysis of 2000 or more colonies. Furthermore, colonies containing the same DNA fragments may be selected for the analysis; this requires colonies in the number of several times 2,000, namely, close to 10,000 colonies to be selected for analysis.
According to conventional methods, base sequencing of the human genome and other organisms, requires digesting of a great length of DNA by restriction enzymes and subcloning the digested DNA into vectors such as plasmids and yeast chromosomes, transforming an appropriate host cell with said plasmids or yeast chromosomes thereby forming a colony derived from one clone containing a single DNA digested product and selecting each DNA fragment by picking the colony. Then DNA fragments are amplified by culturing microbes obtained from each colony to produce a large amounts of the DNA fragment required for base sequencing techniques. Called "cloning or subcloning", this biological technique has disadvantages of taking a lot of time for culturing and is not suitable for automation. Such conventional techniques are discussed in Molecular Cloning, A Laboratory Manual (2nd Version) (Chapters 1 to 4 and Chapter 9, Cold spring Harbor Laboratory Press, 1989).
According to the above techniques, single DNA fragment species required for sequencing are prepared using vectors and transformed colibacillus and yeast requiring the use of a special P2 facility. Another disadvantage of the method is the amount of time required to culture the microbes and to pick large numbers of colonies for analysis, making the method unfit for automation.
Primer Walking (Science, Vol. 258, pages 1787 to 1791, 1992) is known as a method for analysis of long DNA fragments; it provides for sequencing of 40 DNA bases from one end of a fragment using a first primer. A second sequencing step is carried out with a second primer which is an oligomer having a sequence in the vicinity of the first sequenced DNA terminal. Sequencing is carried out step-by-step using new primers which have sequences complementary to the sequenced DNA. Analysis of 1 megabase of DNA requires 2500 sequencing steps with this method and each analysis requires one or more days; thus several years have been required for all analyses.
Thus, such large scale DNA sequencing methods comprise processes requiring much cost and time. Depending on the sizes and types of the DNA fragments digested by restriction enzymes, some of the DNA fragments have been difficult to introduce into vectors by cloning. Consequently, large scale DNA sequencing has been a problem requiring the development of a new technique.